Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases

Abstract

A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251-kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologs in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O2 and methane, in the case of sMMO, to the reduced diiron center for O2 activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of “Fe2O2” intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe2O2 intermediates by blocking reductase binding during O2 activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze very similar reactions as the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic prot...

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

“…It has recently been established that the MMOB modulates the activity of sMMO by inducing conformational changes in the protein, thus allowing the access of dioxygen and methane to the diiron center (through a series of hydrophobic cavities) and restricting proton access to this diiron center during the formation of Fe2O2 intermediates. Moreover, the competition between MMOB and MMOR inhibits undesired electron transfer to the Fe2O2 intermediates, by blocking the binding of MMOR to MMOH during O2 activation [26,27].…”

The concept of photochemical enzyme models – State of the art

“…Although oxidoreductases capable of direct interaction with O 2 in its triplet ground state have been identified [85], the majority of native oxidase enzymes requires additional organic cofactors or redox-active transition metal centers Molecular oxygen from air is a clean, abundant and environmentally benign reagent ideally suited for substrate oxidation processes including selective C-H bond functionalization. Therefore the utilization of oxidase-like reactivity for green synthetic processes based on O 2 or H 2 O 2 is a highly desirable research goal [92][93][94].…”

“…The unique chemistry of high-valent metal oxo-species such as ferryl Fe IV =O intermediates and Mn IV -oxyl radicals plays a prominent role in some of the most difficult to achieve bioinorganic redox processes like selective alkane functionalization and photosynthetic water oxidation [92,[103][104][105][106][107]. With the help of reactive oxo and peroxo species, oxygenase enzymes can catalyze the transfer of one or two O-atoms originating from dioxygen to their substrates.…”

“…Many oxygenases are therefore also investigated as attractive candidates for applications in biotechnology and organic synthesis [108]. Typical examples of oxidase metalloproteins are the soluble and particulate methane monooxygenases (EC 1.14.13.25, EC 1.14.18.3) [92,109], chloroperoxidase (EC 1.11.1.10) [110], nitric oxide synthase (EC 1.14.1.13.39) [111] and aromatic peroxygenase (EC 1.11.2.1) [112]. Among the most widely studied representatives of this class of redox enzymes with catalytically active metal-oxo intermediates are the heme-thiolate monooxygenases of the cytochrome P450 (EC.1.14.14.1) superfamily [113].…”

A novel NADPH-dependent reductase of Sulfobacillus acidophilus TPY phenol hydroxylase: expression, characterization, and functional analysis

“…Although the carboxylatebridged dinuclear iron center is in the hydroxylase component, many reports have demonstrated that efficient hydroxylation of substrates by BMMs is not possible without both the reductase and regulatory components, which physically interact with the hydroxylase component to modulate the hydroxylation activity (Liu et al 1997;Sazinsky and Lippard 2006;Tinberg et al 2011). The regulatory protein couples oxidation of NAD(P)H to hydroxylation of the organic substrate, a process that has been characterized in sMMO and PH (Qian et al 1997;Shinohara et al 1998;Wang et al 2015). The reductase component is usually an flavin adenine dinucleotide (FAD)-and [2Fe-2S]-containing enzyme that transfers electrons from NAD(P)H to the di-iron sites of the terminal oxygenase via the [2Fe-2S] cluster and flavin prosthetic group.…”